This invention relates to protein scaffolds useful, for example, for the generation of products having novel binding characteristics.
Proteins having relatively defined three-dimensional structures, commonly referred to as protein scaffolds, may be used as reagents for the design of engineered products. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected. One particular area in which such scaffolds are useful is the field of antibody mimetic design.
While therapeutic antibodies are known with some successful examples on the market (HERCEPTIN®, AVASTIN®, SYNAGIS®), there is a growing interest in generating antibody fragments as therapeutic proteins. The advantages are the ease of manipulation by molecular biology techniques in order to obtain desired binding characteristics, the ability to express such fragments in microbial systems, and the expectation that antibody fragments will have better tissue penetration than full-length antibodies. One example is REOPRO®.
In addition, there have been efforts to develop small, non-antibody therapeutics, i.e., antibody mimetics, in order to capitalize on the advantages of antibodies and antibody fragments, such as high affinity binding of targets and low immunogenicity and toxicity, while avoiding some of the shortfalls, such as the requirement for intradomain disulfide bonds which require proper refolding, and the tendency for antibody fragments to aggregate and be less stable than full-length IgGs. One example is a “minibody” scaffold, which is related to the immunoglobulin fold, which is designed by deleting three beta strands from a heavy chain variable domain of a monoclonal antibody (Tramontano et al., J. Mol. Recognit. 7:9, 1994). This protein includes 61 residues and can be used to present two hypervariable loops, much like complementarity determining regions (CDRs) in antibodies. These two loops have been randomized and products selected for antigen binding, but thus far the framework appears to have somewhat limited utility due to solubility problems. Another framework used to display loops has been tendamistat, a small protein inhibitor of α-amylase, which contains a 74 residue, six-strand beta sheet sandwich held together by two disulfide bonds and forms 3 CDR-like loops (McConnell and Hoess, J. Mol. Biol. 250:460, 1995).
Other proteins have been tested as frameworks and have been used to display randomized residues on alpha helical surfaces (Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Protein Eng. 8:601, 1995), loops between alpha helices in alpha helix bundles (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by disulfide bridges, such as those of the small protease inhibitors (Markland et al., Biochemistry 35:8045, 1996; Markland et al., Biochemistry 35:8058, 1996; Rottgen and Collins, Gene 164:243, 1995; Wang et al., J. Biol. Chem. 270:12250, 1995).
Thus, there is a need to develop small, stable, artificial antibody-like molecules for a variety of therapeutic and diagnostic applications.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.